Second harmonic imaging nanoprobes and techniques for use thereof

ABSTRACT

Second harmonic nanoprobes for imaging biological samples and a method of using such probes to monitor the dynamics of biological process using a field resonance enhanced second harmonic (FRESH) technique are provided. The second harmonic generating (SHG) nanoprobes are comprised of various kinds of nanocrystals that do not possess an inversion symmetry and therefore are capable of generating second harmonic signals that can then be detected by conventional two-photon microscopy for in vivo imaging of biological processes and structures such as cell signaling, neuroimaging, protein conformation probing, DNA conformation probing, gene transcription, virus infection and replication in cells, protein dynamics, tumor imaging and cancer therapy evaluation and diagnosis as well as quantification in optical imaging.

CROSS-REFERENCE TO RELATED APPLICATIONS

The current application claims priority to U.S. Provisional ApplicationNo. 60/860,439, filed Nov. 21, 2006, and U.S. Provisional ApplicationNo. 60/936,043, filed Jun. 18, 2007, the disclosures of which areincorporated herein by reference.

STATEMENT OF FEDERAL RIGHTS

The U.S. Government has certain rights in this invention pursuant toGrant No. HR0011-04-1-0032 awarded by DARPA.

FIELD OF THE INVENTION

The current invention is directed to a methodology for imagingbiological samples using second harmonic generating nanoprobes.

BACKGROUND OF THE INVENTION

One of the grand open challenges in modern science is to identify cellsor probe molecules and understand the mechanism and dynamics ofbiological processes at the molecular level with high sensitivity andspatiotemporal resolution, and particularly inside living cells andtissue or liquid. As a result of the wealth of information potentiallyaccessible from such biological targets, there has been a growing demandfor imaging tools for biomedical research and medicine. This researchhas led to the development of new techniques like magnetic resonanceimaging (MRI), ultrasound, positron emission tomography (PET), andoptical coherence tomography (OCT). However, these techniques requirehigh costs and some fundamental technological barriers hinder theirwidespread use.

Optical imaging is a recent technique that utilizes photons as aninformation source with applications in a wide range of basic scienceand clinical studies like pharmacology, cellular biology, anddiagnostics. For example, semiconductor nanocrystals, small organic dyesor fluorescent proteins are commonly used as optical. labels in in vivooptical imaging. (See, e.g., X. Michalet et al.,i Science 307, 538 (Jan28, 2005); B. Dubertret et al., Science 298, 1759 (Nov. 29, 2002); M. K.So, C. Xu, A. M. Loening, S. S. Gambhir, J. Rao, Nat Biotechnol 24, 339(March, 2006); N. C. Shaner, P. A. Steinbach, R. Y. Tsien, Nat Methods2, 905 (December, 2005); and B. N. Giepmans, S. R. Adams, M. H.Ellisman, R. Y. Tsien, Science 312, 217 (Apr. 14, 2006), the disclosuresof which are incorporated herein by reference.) Indeed, recent advancesin fluorescence microscopy alone have profoundly changed how cell andmolecular biology is studied in almost every aspect. (For example, see,Lichtman, J. W. & Conchello, J. A. Nat. Methods 2, 910-919 (2005);Michalet, X. et al. Annu. Rev. Biophys. Biomolec. Struct. 32, 161-182(2003); Jares-Erijman, E. A. & Jovin, T. M. Nat. Biotechnol. 21,1387-1395 (2003); Bastiaens, P. I. H. & Squire, A., Trends Cell Biol. 9,48-52 (1999); and Suhling, K., et al, Photochem. Photobiol. Sci. 4,13-22 (2005), the disclosures of which are incorporated herein byreference.)

However, the ultimate need of characterizing biological targets islargely unmet due to fundamental deficiencies associated with the use offluorescent agents. For example, fluorescent probes face two majorlimitations that have a significant impact on the signal strength: 1)dye saturation, because the number of photons emitted by the fluorophorein a given time is restricted by the excited state lifetime, and 2) dyebleaching, which limits the total number of photons per dye. Inaddition, autofluorescence from tissue organic components due toillumination absorption can severely limit the signal-to-noise ratio.Finally, fluorescence is fundamentally an optically incoherent process,and as a result extracting 3D information from the source is inherentlydifficult.

Accordingly, a need exists for a new probe for imaging/detectingbiological structures and processes that avoids the inherenttechnological limitations found in the fluorescent imaging techniques ofthe prior art.

SUMMARY OF THE INVENTION

The current invention is directed to nanoprobes for imaging/detectingstructures and biological processes based on a novel second harmonic(SH) technique.

In one embodiment, the probe nanostructures that generate secondharmonic signals emit coherent waves for imaging biological structureswithout bleaching, blinking or saturation.

In another embodiment, the probe nanostructures are attached tomolecules of interest or delivered to cells of interest.

In another embodiment, the probes of the current invention are formed oftwo dissimilar types of nanostructures: a first exciter nanostructurethat resonates at the frequency of the pump, and a second probenanostructure that generates second harmonic signals.

In still another embodiment the first exciter nanostructure is a metalnanostructure and the second probe nanostructure is a nanocrystal.

In yet another embodiment, the exciter nanostructure is chosen such thatwhen pumped via a continuous wave, modulated or pump source it enhancesthe electric field within a few nanometers of its vicinity. In such anembodiment, the enhanced local field can couple with neighboring probes,and the short-range interactions can then be used as a nanometersensitive distance gauge.

In still yet another embodiment, the current invention is directed to amethod of imaging/detecting with superb sensitivity and spatiotemporalresolution biological process and structures using a field resonanceenhanced second harmonic (FRESH) technique.

In still yet another embodiment, the current invention is directed to amethod of using the SH nanoprobes in a detection scheme, such as, therapid detection of a specific target, the imaging/detection of medicalconditions or neoplasm, and the detection or tracking of a therapeuticagent.

BRIEF DESCRIPTION OF THE DRAWINGS

The description and claims of the current invention will be more fullyunderstood with reference to the following figures and data graphs,which are presented as exemplary embodiments of the invention and shouldnot be construed as a complete recitation of the scope of the invention,wherein:

FIGS. 1 a & 1 b provide emission profiles of an exemplary secondharmonic generation nanocrystal probe in accordance with the currentinvention;

FIG. 2 provides a schematic diagram comparing the properties of thesecond harmonic technique of the current invention with the fluorescenceused in conventional optical systems;

FIG. 3 provides a set of data graphs showing the comparative bleachingand blinking properties for an exemplary second harmonic generationnanocrystal probe in accordance with the current invention and aconventional quantum dot;

FIG. 4 provides a set of data graphs showing the saturation propertiesfor an exemplary second harmonic generation nanocrystal probe inaccordance with the current invention and a conventional quantum dot;

FIGS. 5 a to 5 d provide photographs exhibiting the in vivo imagingproperties of an exemplary second harmonic generation nanocrystal probein accordance with the current invention;

FIG. 6 provides a schematic diagram of the principles of operation of anexemplary embodiment of the second harmonic generation nanocrystal probeimaging technique in accordance with the current invention;

FIG. 7 provides a schematic diagram of the principles of operation of asecond exemplary embodiment of the second harmonic generationnanocrystal probe imaging technique in accordance with the currentinvention; and

FIG. 8 provides a schematic diagram of the principles of a method ofusing the SH nanoprobes in a detection scheme, such as, the rapiddetection of a specific target, the imaging/detection of medicalconditions or neoplasm, and the detection or tracking of a therapeuticagent.

DETAILED DESCRIPTION OF THE INVENTION

The current invention is generally directed to a methodology forimaging/detecting biological samples using nanoprobes capable ofproducing a second harmonic generation response. In addition, theapplication is directed to a technique for dynamic imaging/detectingdynamic processes like molecule conformation changes ormolecule-molecule interactions using a field resonance enhanced secondharmonic technique; referred to from hereinafter as FRESH. Both the SHGnanoprobes and the FRESH imaging technique is designed to overcome theshortcomings of conventional fluorescence-based techniques.

Specifically, to overcome the limitations inherent in conventionalimaging techniques, the current invention is drawn to an imagingmethodology that uses second harmonic generating (SHG) nanoprobes thatare suitable for (in vivo/in vitro) imaging/detecting and can avoid mostof the inherent drawbacks encountered in classical optical systems. Thekey element of this invention is based on labeling molecules oridentifying cells of interest with nonlinear materials, e.g., variouskinds of inorganic and/or organic nanocrystals that do not possess aninversion symmetry and therefore are capable of generating secondharmonic signals.

For typical tissue such emissions might range, for example, from 350 to700 nm, although other wavelengths might be used dependent on thematerial to be imaged. These emissions can then be detected by anyoptical based technique, such as, for example, conventional two-photonmicroscopy (for example, for wavelengths in the range of 350 to 700 nmby tuning the wavelength from 700 to 1400 nm), or continuous wave,modulated, or other pulsed lasers having for example nano, pico, femto,or attosecond timeframes.

As discussed above, the basic principle behind the SH nanoprobes of thecurrent invention is to attach to a molecule of interest a probenanostructure that generates a second harmonic signal or to identifycells or tissue of a living subject (in vivo/in vitro) using such probenanostructures. Such a structure may be any organic, inorganic orcombination of organic and inorganic nanocrystal, such as, for exampleBaTiO₃, SiC, ZnO, ZnS, ZnSe, ZnTe, CdS, CdSe, CdTe, GaAs, GaSb, GaP,GaN, InSb, LiNbO₃, KNbO₃, KTiOPO₄, Fe(IO₃)₃, Au, Ag,N-(4-nitrophenyl)-(L)-prolinol (NPP), urea, 4-Nitroaniline,2-Methyl-4-nitroaniline (MA), 3-Methyl-4-methoxy-4′-nitrostilbene),β-BaB2O4 (Beta-Barium Borate/BBO, LiB3O5 (Lithium Triborate/LBO), LiNbO3(Lithium Niobate/LN), KTiOPO4 (Potassium Titanyl Phosphate/KTP), AgGaS2(Silver Thiogallate/AGS), AgGaSe2 (Silver Gallium Selenide/AGSe), ZnGeP2(Zinc Germanium Phosphide/ZGP), GaSe (Gallium Selenide), KH2PO4(Potassium Dihydrogen Phosphate/KDP), NH4H2PO4 (Ammonium DihydrogenPhosphate (ADP), KD2PO4 (Deuterated Potassium DihydrogenPhosphate/DKDP), CsLiB6O10 (Cesium Lithium Borate/CLBO), KTiOAsO4(Potassium Titanyl Arsenate/KTA), KNbO3 (Potassium Niobate/KN), LiTaO3(Lithium Tantalate/LT), RbTiOAsO4 (Rubidium Titanyl Arsenate/RTA),BaTiO3 (Barium Titanate), MgBaF4 (Magnesium Barium Fluoride), GaAs(Gallium Arsenide), BiB3O6 (Bismuth Triborate/BIBO), K2Al2B2O7(Potassium Aluminum Borate/KABO), KBe2BO3F2 (PotassiumFluoroboratoberyllate/KBBF), BaAlBO3F2 (Barium AluminumFluoroborate/BABF), La2CaB10O19 (Lanthanum Calcium Borate/LCB),GdCa40(BO3)3 (Gadolinium Calcium Oxyborate/GdCOB), YCa40(BO3)3 (YttriumCalcium Oxyborate/YCOB), Li2B4O7 (Lithium Tetraborate/LB4), LiRbB4O7(Lithium Rubidium Tetraborate/LRB4), CdHg(SCN)4 (Cadmium MercuryThiocyanate/CMTC), RbTiOPO4 (Rubidium Titanyl Phosphate/RTP), LiInS2(Lithium Thioindate/LIS), LiInSe2 (Lithium Indium Selenide/LISe),KB5O84H2O (Potassium Pentaborate Tetrahydrate/KB5), CsB3O5 (CesiumTriborate/CBO), C4H7D12N4PO7 (Deuterated L-Arginine PhosphateMonohydrate/DLAP), a-HIO3 (a-Iodic Acid), LiCOOHH2O (Lithium FormateMonohydrate/LFM), CsH2AsO4 (Cesium Dihydrogen Arsenate/CDA), CsD2AsO4(Deuterated Cesium Dihydrogen Arsenate/DCDA), RbH2PO4 (RubidiumDihydrogen Phosphate/RDP), CsTiOAsO4 (Cesium Titanyl Arsenate/CTA),Ba2NaNb5O15 (Barium Sodium Niobate/BNN), K3Li2Nb5O15 (Potassium LithiumNiobate/KLN), CO(NH2)2 (Urea), LiIO3 (Lithium Iodate), Ag3AsS3(Proustite), HgGa2S4 (Mercury Thiogallate), CdGeAs2 (Cadmium GermaniumArsenide/CGA), Ti3AsSe3 (Thallium Arsenic Selenide/TAS), CdSe (CadmiumSelenide), ZnO (Zinc Oxide), ZnS (Zinc Sulfide), ZnSe (Zinc Selenide),ZnTe (Zinc Telluride), CdS (Cadmium Sulfide), SiC (Silicon Carbide), andGaN (Gallium Nitride), GaSb (Gallium Antimonide), among others.

In turn, the molecule of interest may be, for example, a protein, DNA,RNA, a cell or tissue. If the molecule of interest is a cell, such acell may be, for example, a cancer cell, stem cell, or tumor.

Although a few specific examples of possible probe nanostructurenanocrystals are described above, it should be understood that anynanostructure, defined hereinafter as a structure of ≦10 μm, capable ofsecond harmonic generation may be used in the current invention. Therequirement of the materials, as discussed above, being that thenanostructure not possess an inversion symmetry center.

FIG. 1 provides an exemplary data graph for a second harmonic emissionprofile generated from BaTiO₃-nanocrystals in accordance with thecurrent invention. As shown, the second harmonic emission ranges from380 to 485 nm displaying, unlike many other optical probes, discreteemission peaks of around 10 nm, and was generated by conventionaltwo-photon excitation, where the excitation energy ranges from 760 to970 nm.

Although only single types of SH nanoprobes are discussed above, itshould be understood that a plurality of SH nanoparticles displayingdistinct emission profiles can be used to identify various labeledmolecules or cells of interest in parallel. Although one exemplaryexcitation profile generated through standard two-photon excitation isprovided in FIG. 1, it should be understood that any conventionalexcitation source may be used that is compatible with second harmonicgeneration.

Second harmonic generation has many inherent advantages overfluorescence that open the possibility of a wide variety ofapplications. These advantages are discussed with reference to theschematic diagram provided in FIG. 2.

First, as a parametric nonlinear optical process, second harmonicgeneration does not involve real electron energy transition but onlyvirtual transitions. Fluorescence, on the other hand, involves actualenergy transition of electrons. As a result, the response time of secondharmonic generation is at the femtosecond level, about four to fiveorders of magnitude faster than the nanosecond response time offluorescence, allowing very fast and sensitive detection of moleculeswith appropriate detection systems. (See, e.g., R. W. Boyd, Nonlinearoptics (Academic Press, San Diego, Calif., ed. 2nd, 2003), pp. xvii, 578p, the disclosure of which is incorporated herein by reference.)

Second, biological tissue does not often assemble into large, orderednoncentrosymmetric structures. As a result, biological tissue does notgenerate a strong SH signal, therefore, the second harmonic generatingcrystals can be imaged with sharp contrast (high signal-to-noise ratio)when presenting in vivo, allowing detection of single nanocrystalsattached to molecules of interest or identification of cells of interestharboring a nanocrystal in tissue.

Third, unlike fluorescent dyes, second harmonic generating nanocrystalsdo not undergo photo-bleaching or blinking, as shown in FIG. 3. In thisset of data graphs, SHG single BaTiO₃ nanocrystals and CdSe/ZnS quantumdots (QD) were immobilized in 20% polyacrylamide and illuminated 500times within 25 s with 820 nm light. As shown, whereas the QD signalfluctuates displaying blinking and photobleaching, the second harmonicsignal intensity of BaTiO₃ is constant, making it a superior singlemolecule detection probe. (See, e.g., W. Denk, J. H. Strickler, W. W.Webb, Science 248, 73 (Apr. 6, 1990), the disclosure of which isincorporated herein by reference.)

Fourth, again unlike fluorescent dyes, SHG nanocrystals do not undergophoto-saturation with increasing illumination intensity, as shown inFIG. 4. In this set of data graphs, BaTiO₃ nanocrystals and CdSe/ZnSquantum dots (QD) were immobilized in 20% polyacrylamide and illuminatedwith increasing 820 nm light intensity. As shown, signal saturation ofQD occurs already at very low power levels, whereas the second harmonicsignal of BaTiO₃ nanocrystals increases quadratically, allowing veryefficient visualization or detection of, for example, a single moleculeattached to such as a nanoprobe crystal, for example, in tissue orsample solution by simply increasing the illumination power. (See, e.g.,C. K. Sun, Adv Biochem Eng Biotechnol 95, 17 (2005), the disclosure ofwhich is incorporated herein by reference.)

Finally, the SHG nanocrystal probes of the current invention show a highpH stability allowing targeting a wider range of molecules of interest,such as, for example, acidic organelles without signal loss.

The biocompatibility of the technique was also studied, and it has beenshown that injected embryos developed indistinguishably from uninjectedcounterparts.

Although only specific embodiments of the invention are discussed aboveand in the examples below, it should be understood that the uniquecombination of properties possessed by the second harmonic nanoprobes ofthe current invention allows for a number of applications including, forexample; protein, DNA, RNA and tumor imaging and cancer or stem celltherapy evaluation and diagnosis as well as quantification in opticalimaging, (in vivo/in vitro) imaging of biological processes such as cellsignaling, neuroimaging, protein conformation probing, DNA conformationprobing, gene transcription, and virus infection and replication incells. In addition the SHG nanoprobes of the current invention may beused to for a number of (in vivo/in vitro) imaging applications.

EXAMPLE 1 SH Nanoprobe Imaging

To demonstrate the superior imaging properties of the SHG nanocrystalprobes of the current invention, BaTiO3 nanocrystals were injected intozebrafish embryos. Two days after cytoplasmic injection the nanocrystalswere excited with femtosecond pulsed 820 nm light. As shown in thephotographic plates provided in FIGS. 5A to 5D, the low energyexcitation of the probe nanocrystals results in a strong second harmonicsignal. (the bright point of light in the center of the image in FIG.5A). This signal is detectable in epi-mode (FIG. 5A) as well as intrans-mode (FIG. 5B) throughout the whole zebrafish body (nanocrystalindicated at arrowhead in figures), proving the coherence of thetechnique. In contrast, the endogenous second harmonic signal from thetail-muscles can only be detected with relatively high energy excitationin the trans-mode (FIG. 5B). (For discussion, see, e.g., P. J.Campagnola, L. M. Loew, Nat Biotechnol 21, 1356 (November, 2003); and P.J. Campagnola, et al., J Biomed Opt 6, 277 (July, 2001)., thedisclosures of which are incorporated herein by reference.) FIG. 5Cprovides an image developed from injecting a conventional Bodipy TRmethyl ester dye to label the extracellular matrix and cell membranes.FIG. 5D provides a merged picture showing the signal from the inventiveprobe combined with the images of the tissue developed from othertechniques.

This exemplary image shows that the SHG nanocrystal probes of thecurrent invention provide superb signal-to-noise ratio after in vivoinjections allowing detection in rather deep organs, as well as thepotential for real-time biodistribution monitoring. In addition, unlikeendogenous second harmonic generation from ordered, noncentrosymmetricstructures like collagen or myosin, which can only be detected intrans-mode, SHG nanocrystals can be detected both in trans-mode as wellas in epi-mode allowing for the ability to isolate the probe signal fromthe background signal generated by the surrounding biologicalstructures.

EXAMPLE 2 Field Resonance Enhanced Second Harmonic Technique

In addition to simple second harmonic imaging using the second harmonicgenerating nanoprobes of the current invention, the nanoprobes may alsobe used in a field resonance enhanced mode to allow access to a numberof biological processes that can occur below the nanosecond time frame.Using this field resonance enhanced second harmonic (FRESH) technique inaccordance with the current invention it is possible to examine thedynamics of biological processes with high sensitivity andspatiotemporal resolution.

To understand the potential importance of the FRESH technique it isnecessary to examine the inner workings of most biological processes.Besides having highly complex three-dimensional (3D) structures spanninga large range of length scales, living organisms by their nature arevery dynamic: molecular processes such as protein, DNA, and RNAconformations—that take place in a timescale ranging from 100 fs to 100s while the organisms move and metabolize—as well as molecule-moleculeinteractions such as, but not limited to, protein-protein, protein-DNA,and protein-RNA interactions. (See, e.g., Whitesides, G. M., Nat.Biotechnol. 21, 1161-1165 (2003); Williams, S. et al., Biochemistry 35,691-697 (1996); Gilmanshin, R., et al., Proc. Natl. Acad. Sci. U.S.A.94, 3709-3713 (1997); Callender, R. H., et al., Annual Review ofPhysical Chemistry 49, 173-202 (1998); Trifonov, A. et al., Journal ofPhysical Chemistry B 109, 19490-19495 (2005); Cheatham, T. E., Curr.Opin. Struct. Biol. 14, 360-367 (2004); Millar, D. P., Curr. Opin.Struct. Biol. 6, 322-326 (1996); and Brauns, E. B., et al., PhysicalReview Letters 88 (2002), the disclosures of which are incorporatedherein by reference.) The understanding of these processes not only hasfundamental biological significance, but could also enable the treatmentof a host of human diseases. For example, it has been shown that avariety of serious diseases can be directly linked to proteinmisfolding. (See, e.g., Ding, F., et al., J. Biol. Chem. 280,40235-40240 (2005); and Dobson, C. M., Philos. Trans. R. Soc. Lond. B356, 133-145 (2001), the disclosures of which are incorporated herein byreference.)

Fluorescence resonance energy transfer (FRET) and its associatedtechniques have achieved great success in probing molecular activities.(See, e.g., Selvin, P. R. Nat. Struct. Biol. 7, 730-734 (2000);Greulich, K. O., ChemPhysChem 6, 2458-2471 (2005); Peter, M. &Ameer-Beg, S. M. FLIM. Biol. Cell 96, 231-236 (2004); Day, R. N. &Schaufele, F., Mol. Endocrinol. 19, 1675-1686 (2005); Wallrabe, H. &Periasamy, A., Curr. Opin. Biotechnol. 16, 19-27 (2005); Piehler, J.,Curr. Opin. Struct. Biol. 15, 4-14 (2005); Chen, Y., et al.,Differentiation 71, 528-541 (2003); Truong, K. & Ikura, M. Curr. Opin.Struct. Biol. 11, 573-578 (2001); Zal, T. & Gascoigne, N. R., Curr.Opin. Immunol. 16, 418-427 (2004); and Miyawaki, A., Dev. Cell 4,295-305 (2003), the disclosures of which are incorporated herein byreference.) However, these techniques still have the same basiclimitations of fluorescence discussed above, including blinking,bleaching, and saturation, which restricts the sensitivity,signal-to-noise ratio, and spatiotemporal resolution. The integration ofthe nanoprobes of the current invention with a field resonanceenhancement technique in the FRESH protocol set forth herein allows forgreatly improved capabilities for the spatiotemporalvisualization/detection of (single) molecule conformation changes ormolecule-molecule interactions.

The basic principle of the FRESH technique is illustrated in FIG. 6,where a simple DNA hairpin molecule is used as an example. In the FRESHmethodology, two dissimilar types of nanostructures are used to labelmolecules of interest. First, an exciter nanostructure that resonates atthe frequency of the pump is attached to the molecule. Such a structuremay be a metal nanostructure, such as, for example, Au-nanorods,Au-nanospheres or Au-nanoshells, and other materials as well. Second, aprobe nanostructure that generates a second harmonic signal inaccordance with the current invention is attached to the molecule. Asbefore such a probe nanostructure can be any construct organic,inorganic or a combination thereof that does not possess an inversionsymmetry.

It should also be understood that although a simple DNA hairpinstructure is shown as an example in FIG. 6, the FRESH methodology of thecurrent invention is equally applicable to other biological processesand structures, such as, for example, protein folding or protein-proteininteraction. Alternatively, the technique may be used as biosensor todetect any phenomenon of interest. FIG. 7, provides a schematic diagramof the operation of such a sensor, the operation of which will bediscussed generically below.

During operation of the SHG probes of the current invention, as shown inFIGS. 6 and 7 above, a continuous wave, modulated or pump energy source,such as a laser emission is focused on a sample of interest. When theexciter nanostructure is energized at the resonance frequency of theenergy source, the exciter enhances the electric field within a fewnanometers of its vicinity. (See, e.g., Averitt, R. D., Westcott, S. L.& Halas, N. J., J. Opt. Soc. Am. B 16, 1824-1832 (1999), the disclosureof which is incorporated herein by reference.) This enhanced local fieldcouples with neighboring probes when brought within a few nanometers ofa probe nanostructure. The coupling of the pump nanostructure with theprobe nanostructure enhances the second harmonic emission from the probenanostructure by orders of magnitude allowing for detection of thesecond harmonic emission. Similar effects have been demonstrated inSurface Enhanced Raman Spectroscopy (SERS) with an enhancement factor of10¹⁴. (See, e.g., Nie, S. M. & Emery, S. R., Science 275, 1102-1106(1997), the disclosure of which is incorporated herein by reference.)The enhanced electric field around the resonant exciter can serve as aruler of nanometer resolution enabling high resolution imaging. TheFRESH technique also allows for the (in vivo/in vitro) imaging ofbiological processes such as cell signaling, neuroimaging, proteinconformation probing, DNA conformation probing, gene transcription, andvirus infection and replication in cells in real time.

EXAMPLE 3 SH Nanoprobe Rapid Detection System

The present invention also relates to the use of SH nanoprobes in rapiddetection systems, as shown in FIG. 8, that can be used by primary carelevel practitioners and field workers in hospitals or doctor's officesin the detection of disorders such as, for example, infectious diseaseor cancer from liquid or solid sources, among others. Because of thewide applicability of the SH nanoprobes of the current invention, SHnanoprobe detection mixtures can be designed for detection of a varietyof conditions, and because of their high sensitivity such detectionmixtures require only minimal amounts of SH nanoprobes for eachanalysis. Indeed, even the presence of a single sign of pathogenesisincluding, for example, an antigen, peptide sequence, nucleic acidsequence, RNA or DNA of an infectious pathogen could be detected usingthe rapid detection system of the current invention. As a result, thesesystems/kits will allow for the detection of a variety of illnesses withportable, inexpensive and easy to use tools, and will allow primary carelevel and field level workers, such as physicians, nurses, and aidworkers to screen and detect a variety of conditions, leading to earlydetection and more prompt treatment than otherwise possible.

In one exemplary embodiment, such a detection method would include thesteps of:

-   -   adding a plurality of SH nanoprobes to the sample;    -   waiting for a period of time to allow the SH nanoprobes to bind        to the substance in the sample to form an SH nanoprobe-substance        mixture;    -   removing SH nanoprobes that do not bind to the substance in the        sample;    -   illuminating the SH nanoprobes-substance mixture in the sample        with any conventional excitation source that is compatible with        second harmonic generation;    -   collecting light returned from the illuminated SH        nanoprobe-substance mixture in the sample;    -   obtaining an image and/or spectrum of the SH nanoprobe substance        mixture in the sample from the collected SH signal; and    -   detecting the substance from the image and/or spectrum of the SH        nanoprobe-substance mixture.

Although as discussed previously attachment of the SH nanoprobe to thetarget of interest is not always necessary, in one embodiment of therapid detection system of the current invention a plurality of opticalSH nanoprobes are conjugated with a predetermined chemical compound. Thepredetermined chemical compound can comprise any target of interest,including, for example a protein, a peptide, a nucleic acid, or anantibody.

As indicated, the sample in such a system may comprise a liquid sample,such as, for example, cerebrospinal fluid, thoracentesis, paracentesis,saliva, urine, semen, mucous, blood, and bronchoalveolar specimen of aliving subject, or in another aspect, the sample may comprise a solidsample such as, for example, stool, pap smear, and buccal membranescrapings of a living subject. In turn, the target of interest mayinclude any substance indicative of pathogenesis including, for example,an antigen, peptide sequence, nucleic acid sequence, RNA or DNA of aninfectious pathogen such as, for example, viral, bacterial, parasiticpathogen, or cancer cells. The pathogenesis itself may correspond to anydisease compatible with such a detection technique, such as, forexample, a disease related to infection, inflammation, or cancer, etc.

The system for detecting a target of interest in a sample includes aplurality of SH nanoprobes, a spectra containing a plurality ofspectrum, each spectrum corresponding to a corresponding optical SHnanoprobe-substance mixture, and an optical device for obtaining animage and/or spectrum of an SH nanoprobe-substance mixture in a sample.In operation, a substance is identifiable from comparison the imageand/or spectrum of the SH nanoprobes-substance mixture in the samplewith the spectra.

As indicated above, the invention comprises the steps of finding a signof pathogenesis from the image and/or spectrum of the SHnanoprobe-substance mixture in the sample and identifying acorresponding disease from the identified sign of pathogenesis of theplurality of SH nanoprobes with a predetermined chemical compound. Suchidentification requires first collecting a signal, then obtaining aspectrum or image, and then identifying a sign of pathogenesis. Althoughany suitable collection of detector, recorder and analyzer may be usedto carry out these steps, some exemplary embodiments are describedbelow.

First, with regard to the step of collecting the signal, any suitabledetector capable of collecting the SH signal returned from theilluminated SH nanoprobe-substance mixture may be used, such as, forexample, an optical spectroscopic system or filter system. Likewise,with regard to the step of obtaining an image and/or spectrum of theilluminated SH nanoprobe-substance mixture from the collected signal,any suitable device capable of recording the signal and obtaining animage or spectrum therefrom may be used. Finally, with regard to thestep of identifying the peak of the SH nanoprobe-substance mixture inthe spectrum at a predetermined wavelength and using the peak of the SHnanoprobe-substance mixture at the predetermined wavelength as the signof pathogenesis to identify the disease may be performed, any suitablesystem, including, for example, an analyzer or data analysis softwaremay be used.

Although not explicitly discussed above as an extension of thisdetection method, the FRESH technique can also be applied to enhance theSH signal.

EXAMPLE 4 Imaging/Detecting Medical Conditions or Neoplasm

In another exemplary embodiment, the SH nanoprobes of the currentinvention may also be used to identify the cause for a medical conditionin a living subject. In such an embodiment, the detection method wouldfurther include the step of conjugating a plurality of SH nanoprobeswith a target responsive to the medical condition, such as, for example,an antibody. Any medical condition compatible with the SH nanoprobes ofthe current invention may be interrogated in this manner. For example,medical conditions related to infection, inflammation, apoptosis,senescence, etc. may thus be identified.

An effective amount of the SH nanoprobes with the target can bedelivered by any suitable means, such as, for example to the circulationsystem, aerodigestive and genitourinary tracts of the living subject.The SH nanoprobes can then be optically imaged or detected withspectroscopy methods or any other imaging method compatible with the SHsignal and the cause of the medical condition can be determined from theimages/spectra of the SH nanoprobe with the target. In one alternativeembodiment, a plurality of SH nanoprobes displaying distinct emissionprofiles can be used to identify various medical conditions in parallel.

In another aspect, the present invention can also be used as a method ofimaging/detecting a living subject for image guided/navigated surgery.In such a method, an effective amount of SH nanoprobes, conjugated withtarget molecules responsive to the surgical target itself can bepreoperatively delivered to surgical area of interest. For exampleantibodies responsive to neoplasm can be delivered to tissue(s)associated with the tumor.

Any target compatible with the SH nanoprobes may be imaged using such asystem. For example, a neoplasm might comprise a tumor (for examplebrain tumor, cervical carcinoma) and/or other abnormal tissues.Likewise, the imaging technique may be used with any compatible surgicalprocedure, such as, for example a biopsy, a surgical excision, a laserablation, etc. The SH nanoprobes can then be detected in the subjectwith any conventional excitation source that is compatible with secondharmonic generation.

Using such an imaging method, a surgical procedure on the tumor can beguided with the detected SH nanoprobes. The method can also be used forimaging the SH nanoprobes post-operatively for assessing the performanceof the surgical procedure.

EXAMPLE 5 Detection or Tracking of Therapeutic Agents

Finally, the present invention also relates to a method for detectingthe delivery of a therapeutic agent. In such an embodiment, thetherapeutic agent would be labeled with an effective amount of SHnanoprobes and delivered to a targeted site of the living subject. Thetargeted site of the living subject would then be imaged with anyconventional excitation source that is compatible with second harmonicgeneration, and the delivery of the therapeutic agent would be detectedfrom the image of the targeted site of the living subject.

The therapeutic agent can comprise any molecule compatible with the SHnanoprobes of the current invention. For example, in one embodiment atherapeutic cell might be used. In such an embodiment any suitabletherapeutic cell might be used including, for example stem cells,package delivery cells carrying a packaged gene or protein, andimmunotherapeutic cells. The labeling for a cell would comprise labelingthe therapeutic cell by delivering an effective amount of SH nanoprobesto the cell. In another example, the therapeutic agent might comprise atherapeutic gene. In such an embodiment the labeling step would compriselabeling the therapeutic gene by conjugating the SH nanoprobe to thetherapeutic gene.

Regardless of the therapeutic agent chosen, the delivery step wouldcomprise delivering the therapeutic agent labeled with the SH nanoprobesby any suitable means, such as, for example intravenous injection.

SUMMARY

While the above description contains many specific embodiments of theinvention, these should not be construed as limitations on the scope ofthe invention, but rather as an example of one embodiment thereof. Forexample, the SHG probes can be used in conjunction with detectiontechniques other than the disclosed laser techniques. In addition, theapplications of the SHG probes in accordance with embodiments of theinvention are not limited to the FRESH dynamic monitoring techniquethose described above and can be any kind of biological or other imagingapplications in which the SHG nanoprobes may be functionally attached tothe target molecule(s). Indeed, it is not necessarily even essentialthat the nanoprobes of the current invention be attached to themolecules of interest. For example, the nanoprobes could be used toperform cell lineage analysis, which can be performed without attachingthe crystals to the proteins. Accordingly, the scope of the inventionshould be determined not by the embodiments illustrated, but by theappended claims and their equivalents.

1. A second harmonic generating nanoprobe system comprising: a probenanostructure, having no inversion symmetry such that it generates asecond harmonic emission when radiated by an external excitation source;and an external excitation source for radiating the probe nanostructureat a frequency such that the probe nanostructure generates a secondharmonic emission.
 2. The second harmonic nanoprobe system of claim 1,wherein the probe nanostructure is a nanocrystal selected from the groupconsisting of organic, inorganic and combinations thereof.
 3. The secondharmonic nanoprobe system of claim 2, wherein the nanocrystal isselected from the group consisting of BaTiO₃, SiC, ZnO, ZnS, ZnSe, ZnTe,CdS, CdSe, CdTe, GaAs, GaSb, GaP, GaN, InSb, LiNbO₃, KNbO₃, KTiOPO₄,Fe(IO₃)₃, Au, Ag, N-(4-nitrophenyl)-(L)-prolinol (NPP), urea,4-Nitroaniline, 2-Methyl-4-nitroaniline (MNA),3-Methyl-4-methoxy-4′-nitrostilbene), β-BaB2O4 (Beta-Barium Borate/BBO,LiB3O5 (Lithium Triborate/LBO), LiNbO3 (Lithium Niobate/LN), KTiOPO4(Potassium Titanyl Phosphate/KTP), AgGaS2 (Silver Thiogallate/AGS),AgGaSe2 (Silver Gallium Selenide/AGSe), ZnGeP2 (Zinc GermaniumPhosphide/ZGP), GaSe (Gallium Selenide), KH2PO4 (Potassium DihydrogenPhosphate/KDP), NH4H2PO4 (Ammonium Dihydrogen Phosphate (ADP), KD2PO4(Deuterated Potassium Dihydrogen Phosphate/DKDP), CsLiB6O10 (CesiumLithium Borate/CLBO), KTiOAsO4 (Potassium Titanyl Arsenate/KTA), KNbO3(Potassium Niobate/KN), LiTaO3 (Lithium Tantalate/LT), RbTiOAsO4(Rubidium Titanyl Arsenate/RTA), BaTiO3 (Barium Titanate), MgBaF4(Magnesium Barium Fluoride), GaAs (Gallium Arsenide), BiB3O6 (BismuthTriborate/BIBO), K2Al2B2O7 (Potassium Aluminum Borate/KABO), KBe2BO3F2(Potassium Fluoroboratoberyllate/KBBF), BaAlBO3F2 (Barium AluminumFluoroborate/BABF), La2CaB10O19 (Lanthanum Calcium Borate/LCB),GdCa40(BO3)3 (Gadolinium Calcium Oxyborate/GdCOB), YCa4O(BO3)3 (YttriumCalcium Oxyborate/YCOB), Li2B4O7 (Lithium Tetraborate/LB4), LiRbB4O7(Lithium Rubidium Tetraborate/LRB4), CdHg(SCN)4 (Cadmium MercuryThiocyanate/CMTC), RbTiOPO4 (Rubidium Titanyl Phosphate/RTP), LiInS2(Lithium Thioindate/LIS), LiInSe2 (Lithium Indium Selenide/LISe),KB5O84H2O (Potassium Pentaborate Tetrahydrate/KB5), CsB3O5 (CesiumTriborate/CBO), C4H7D12N4PO7 (Deuterated L-Arginine PhosphateMonohydrate/DLAP), a-HIO3 (a-Iodic Acid), LiCOOHH2O (Lithium FormateMonohydrate/LFM), CsH2AsO4 (Cesium Dihydrogen Arsenate/CDA), CsD2AsO4(Deuterated Cesium Dihydrogen Arsenate/DCDA), RbH2PO4 (RubidiumDihydrogen Phosphate/RDP), CsTiOAsO4 (Cesium Titanyl Arsenate/CTA),Ba2NaNb5O15 (Barium Sodium Niobate/BNN), K3Li2Nb5O15 (Potassium LithiumNiobate/KLN), CO(NH2)2 (Urea), LiIO3 (Lithium Iodate), Ag3AsS3(Proustite), HgGa2S4 (Mercury Thiogallate), CdGeAs2 (Cadmium GermaniumArsenide/CGA), Ti3AsSe3 (Thallium Arsenic Selenide/TAS), CdSe (CadmiumSelenide), ZnO (Zinc Oxide), ZnS (Zinc Sulfide), ZnSe (Zinc Selenide),ZnTe (Zinc Telluride), CdS (Cadmium Sulfide), SiC (Silicon Carbide), GaN(Gallium Nitride), and GaSb (Gallium Antimonide).
 4. The second harmonicnanoprobe system of claim 1, wherein the probe nanostructure is lessthan or equal to 10 μm.
 5. The second harmonic nanoprobe system of claim1, wherein the probe nanostructure is one of either attached to orintroduced into a target of interest.
 6. The second harmonic nanoprobesystem of claim 1, wherein the excitation source is selected from thegroup consisting of continuous wave, modulated and pulsed lasers.
 7. Afield resonance enhanced second harmonic system comprising: an exciternanostructure, said exciter nanostructure designed to produce anenhanced local electrical field of a specified frequency when exposed toan excitation source; a probe nanostructure, said probe nanostructurehaving no inversion symmetry such that it generates a second harmonicresonance emission when brought within range of the resonant electricalfield of said exciter nanostructure; and an external excitation sourcefor radiating the exciter nanostructure at a frequency such that theexciter nanostructure generates an enhanced electrical field.
 8. Thefield resonance enhanced second harmonic system of claim 7, wherein theexciter nanostructure is a metal nanostructure.
 9. The field resonanceenhanced second harmonic system of claim 8, wherein the metalnanostructure is a nanostructure selected from the group consisting ofnanorods, nanospheres or nanoshells.
 10. The field resonance enhancedsecond harmonic system of claim 9, wherein the metal nanostructure ismade of gold, silver, copper, aluminum, palladium, or platinum.
 11. Thefield resonance enhanced second harmonic system of claim 7, wherein theprobe nanostructure is a nanocrystal selected from the group consistingof organic, inorganic and combinations thereof.
 12. The field resonanceenhanced second harmonic system of claim 11, wherein the nanocrystal isselected from the group consisting of of BaTiO₃, SiC, ZnO, ZnS, ZnSe,ZnTe, CdS, CdSe, CdTe, GaAs, GaSb, GaP, GaN, InSb, LiNbO₃, KNbO₃,KTiOPO₄, Fe(IO₃)₃, Au, Ag, N-(4-nitrophenyl)-(L)-prolinol (NPP), urea,4-Nitroaniline, 2-Methyl-4-nitroaniline (MNA),3-Methyl-4-methoxy-4′-nitrostilbene), β-BaB2O4 (Beta-Barium Borate/BBO,LiB3O5 (Lithium Triborate/LBO), LiNbO3 (Lithium Niobate/LN), KTiOPO4(Potassium Titanyl Phosphate/KTP), AgGaS2 (Silver Thiogallate/AGS),AgGaSe2 (Silver Gallium Selenide/AGSe), ZnGeP2 (Zinc GermaniumPhosphide/ZGP), GaSe (Gallium Selenide), KH2PO4 (Potassium DihydrogenPhosphate/KDP), NH4H2PO4 (Ammonium Dihydrogen Phosphate (ADP), KD2PO4(Deuterated Potassium Dihydrogen Phosphate/DKDP), CsLiB6O10 (CesiumLithium Borate/CLBO), KTiOAsO4 (Potassium Titanyl Arsenate/KTA), KNbO3(Potassium Niobate/KN), LiTaO3 (Lithium Tantalate/LT), RbTiOAsO4(Rubidium Titanyl Arsenate/RTA), BaTiO3 (Barium Titanate), MgBaF4(Magnesium Barium Fluoride), GaAs (Gallium Arsenide), BiB3O6 (BismuthTriborate/BIBO), K2Al2B2O7 (Potassium Aluminum Borate/KABO), KBe2BO3F2(Potassium Fluoroboratoberyllate/KBBF), BaAlBO3F2 (Barium AluminumFluoroborate/BABF), La2CaB10O19 (Lanthanum Calcium Borate/LCB),GdCa4O(BO3)3 (Gadolinium Calcium Oxyborate/GdCOB), YCa4O(BO3)3 (YttriumCalcium Oxyborate/YCOB), Li2B4O7 (Lithium Tetraborate/LB4), LiRbB4O7(Lithium Rubidium Tetraborate/LRB4), CdHg(SCN)4 (Cadmium MercuryThiocyanate/CMTC), RbTiOPO4 (Rubidium Titanyl Phosphate/RTP), LiInS2(Lithium Thioindate/LIS), LiInSe2 (Lithium Indium Selenide/LISe),KB5O84H2O (Potassium Pentaborate Tetrahydrate/KB5), CsB3O5 (CesiumTriborate/CBO), C4H7D12N4PO7 (Deuterated L-Arginine PhosphateMonohydrate/DLAP), a-HIO3 (a-Iodic Acid), LiCOOHH2O (Lithium FormateMonohydrate/LFM), CsH2AsO4 (Cesium Dihydrogen Arsenate/CDA), CsD2AsO4(Deuterated Cesium Dihydrogen Arsenate/DCDA), RbH2PO4 (RubidiumDihydrogen Phosphate/RDP), CsTiOAsO4 (Cesium Titanyl Arsenate/CTA),Ba2NaNb5O15 (Barium Sodium Niobate/BNN), K3Li2Nb5O15 (Potassium LithiumNiobate/KLN), CO(NH2)2 (Urea), LiIO3 (Lithium Iodate), Ag3AsS3(Proustite), HgGa2S4 (Mercury Thiogallate), CdGeAs2 (Cadmium GermaniumArsenide/CGA), Ti3AsSe3 (Thallium Arsenic Selenide/TAS), CdSe (CadmiumSelenide), ZnO (Zinc Oxide), ZnS (Zinc Sulfide), ZnSe (Zinc Selenide),ZnTe (Zinc Telluride), CdS (Cadmium Sulfide), SiC (Silicon Carbide), andGaN (Gallium Nitride), GaSb (Gallium Antimonide).
 13. The fieldresonance enhanced second harmonic system of claim 7, wherein the probenanostructure is less than or equal to 10 μm.
 14. The field resonanceenhanced second harmonic system of claim 7, wherein the probenanostructure and the emitter nanostructure are both attached to asingle target of interest.
 15. The field resonance enhanced secondharmonic system of claim 7, wherein the excitation source is selectedfrom the group consisting of continuous wave, modulated and pulsedlasers.
 16. A method of probing structures and biological processescomprising: interspersing a probe nanostructure with a target ofinterest, such that the probe nanostructure interacts with the target ofinterest, and wherein the probe nanostructure has no inversion symmetrysuch that it generates a second harmonic emission when radiated by anexcitation source; radiating the probe nanostructure with an externalexcitation source; and detecting the second harmonic emission from thenanoprobe.
 17. The method of claim 16, wherein the probe nanostructureis a nanocrystal selected from the group consisting of organic,inorganic and combinations thereof.
 18. The method of claim 16, whereinthe target of interest is selected from the group consisting of anantigen, a peptide sequence, a nucleic acid sequence, RNA and DNA. 19.The method of claim 16, wherein the target of interest is indicative ofthe presence of one of either a specific medical condition or a specificpathogenesis.
 20. The method of claim 16, wherein the probenanostructure is one of either directly attached to or introduced intothe target of interest.
 21. The method of claim 16, wherein the probenanostructure is conjugated to a probe molecule that is sensitive to thepresence of the target of interest.
 22. The method of claim 16, whereinthe step of radiating is conducted while the probe nanostructure isexternal to the source of the target of interest.
 23. The method ofclaim 16, wherein the step of radiating is conducted while the probenanostructure is in vivo to the source of the target of interest. 24.The method of claim 23, wherein the emissions from the probenanostructure are utilized to image the specific location of the targetof interest within the source of the target of interest.
 25. The methodof claim 16, wherein the target of interest is collected in one ofeither a liquid or solid sample.
 26. The method of claim 16, furthercomprising interspersing an exciter nanostructure to the target ofinterest, said exciter nanostructure designed to produce an enhancedelectrical field of a specified frequency when exposed to an excitationsource, wherein the probe nanostructure generates a second harmonicresonance emission when brought within range of the enhanced electricalfield of said exciter nanostructure.
 27. The method of claim 16, whereinthe excitation source is selected from the group consisting ofcontinuous wave, modulated and pulsed lasers.
 28. A method of fieldresonance enhanced second harmonic detection comprising: attaching anexciter nanostructure to a target of interest, said exciternanostructure designed to produce an enhanced electrical field of aspecified frequency when exposed to an excitation source; attaching aprobe nanostructure to the target of interest, said probe nanostructurehaving no inversion symmetry such that it generates a second harmonicresonance emission when brought within range of the enhanced electricalfield of said exciter nanostructure; radiating the structure of interestwith an external excitation source; and detecting the second harmonicemission from the nanoprobe.
 29. The method of claim 27, wherein theexciter nanostructure is a metal nanostructure selected from the groupconsisting of nanorods, nanospheres or nanoshells.
 30. The method ofclaim 27, wherein the probe nanostructure and the emitter nanostructureare both attached to a single target of interest.
 31. The method ofclaim 27, wherein the probe nanostructure is a nanocrystal selected fromthe group consisting of organic, inorganic and combinations thereof. 32.The method of claim 30, wherein the nanocrystal is selected from thegroup consisting of BaTiO₃, SiC, ZnO, ZnS, ZnSe, ZnTe, CdS, CdSe, CdTe,GaAs, GaSb, GaP, GaN, InSb, LiNbO₃, KNbO₃, KTiOPO₄, Fe(IO₃)₃, Au, Ag,N-(4-nitrophenyl)-(L)-prolinol (NPP), urea, 4-Nitroaniline,2-Methyl-4-nitroaniline (MNA), 3-Methyl-4-methoxy-4′-nitrostilbene),β-BaB2O4 (Beta-Barium Borate/BBO, LiB3O5 (Lithium Triborate/LBO), LiNbO3(Lithium Niobate/LN), KTiOPO4 (Potassium Titanyl Phosphate/KTP), AgGaS2(Silver Thiogallate/AGS), AgGaSe2 (Silver Gallium Selenide/AGSe), ZnGeP2(Zinc Germanium Phosphide/ZGP), GaSe (Gallium Selenide), KH2PO4(Potassium Dihydrogen Phosphate/KDP), NH4H2PO4 (Ammonium DihydrogenPhosphate (ADP), KD2PO4 (Deuterated Potassium DihydrogenPhosphate/DKDP), CsLiB6O10 (Cesium Lithium Borate/CLBO), KTiOAsO4(Potassium Titanyl Arsenate/KTA), KNbO3 (Potassium Niobate/KN), LiTaO3(Lithium Tantalate/LT), RbTiOAsO4 (Rubidium Titanyl Arsenate/RTA),BaTiO3 (Barium Titanate), MgBaF4 (Magnesium Barium Fluoride), GaAs(Gallium Arsenide), BiB3O6 (Bismuth Triborate/BIBO), K2Al2B2O7(Potassium Aluminum Borate/KABO), KBe2BO3F2 (PotassiumFluoroboratoberyllate/KBBF), BaAlBO3F2 (Barium AluminumFluoroborate/BABF), La2CaB10O19 (Lanthanum Calcium Borate/LCB),GdCa4O(BO3)3 (Gadolinium Calcium Oxyborate/GdCOB), YCa4O(BO3)3 (YttriumCalcium Oxyborate/YCOB), Li2B4O7 (Lithium Tetraborate/LB4), LiRbB4O7(Lithium Rubidium Tetraborate/LRB4), CdHg(SCN)4 (Cadmium MercuryThiocyanate/CMTC), RbTiOPO4 (Rubidium Titanyl Phosphate/RTP), LiInS2(Lithium Thioindate/LIS), LiInSe2 (Lithium Indium Selenide/LISe),KB5O84H2O (Potassium Pentaborate Tetrahydrate/KB5), CsB3O5 (CesiumTriborate/CBO), C4H7D12N4PO7 (Deuterated L-Arginine PhosphateMonohydrate/DLAP), a-HIO3 (a-Iodic Acid), LiCOOHH2O (Lithium FormateMonohydrate/LFM), CsH2AsO4 (Cesium Dihydrogen Arsenate/CDA), CsD2AsO4(Deuterated Cesium Dihydrogen Arsenate/DCDA), RbH2PO4 (RubidiumDihydrogen Phosphate/RDP), CsTiOAsO4 (Cesium Titanyl Arsenate/CTA),Ba2NaNb5O15 (Barium Sodium Niobate/BNN), K3Li2Nb5O15 (Potassium LithiumNiobate/KLN), CO(NH2)2 (Urea), LiIO3 (Lithium Iodate), Ag3AsS3(Proustite), HgGa2S4 (Mercury Thiogallate), CdGeAs2 (Cadmium GermaniumArsenide/CGA), Ti3AsSe3 (Thallium Arsenic Selenide/TAS), CdSe (CadmiumSelenide), ZnO (Zinc Oxide), ZnS (Zinc Sulfide), ZnSe (Zinc Selenide),ZnTe (Zinc Telluride), CdS (Cadmium Sulfide), SiC (Silicon Carbide), andGaN (Gallium Nitride), GaSb (Gallium Antimonide).
 33. The method ofclaim 27, wherein the excitation source is selected from the groupconsisting of continuous wave, modulated and pulsed lasers.